Human tumor suppressor polypeptides

ABSTRACT

The present invention provides a human tumor suppressor (TUPRO-2) and polynucleotides which identify and encode TUPRO-2. The invention also provides expression vectors and host cells, agonists, antibodies, or antagonists. The invention provides methods for treating diseases associated with expression of TUPRO-2.

[0001] This application is a continuation application of U.S.application Ser. No. 09/205,749, filed Dec. 4, 1998, which is adivisional application of U.S. application Ser. No. 08/822,262, filedMar. 20, 1997, issued Dec. 15, 1998 as U.S. Pat. No. 5,849,899, entitledHUMAN TUMOR SUPPRESSOR, all of which applications and patents are herebyexpressly incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a novel human tumor suppressor and to the use of these sequences inthe diagnosis, prevention, and treatment of cancer.

BACKGROUND OF THE INVENTION

[0003] When tumorigenic and non-tumorigenic cells are fused in culturethe resulting hybrid cells are usually non-tumorigenic (Knudson, A. G.(1993) Proc. Natl. Acad. Sci. 90: 10914-10921). Heritable factors withinthe non-tumorigenic cell suppress tumor development. Several of thesetumor suppressors, including the retinoblastoma gene and p53, have beencharacterized. Since tumor suppression is of great interest toresearchers and clinicians seeking to control cancer growth, both theretinoblastoma gene and p⁵³ have become candidates for the developmentof cancer therapeutics (Antelman, D. et al. (1995) Oncogene 10: 697-704;Hamada, K. et al. (1996) Cancer Res. 56: 3047-3054). 15 Todd, R. et al.(1995, FASEB J. 9: 1362-1370) recently described a novel tumorsuppressor gene, Doc-1. Doc-1 is structurally altered during oralcarcinogenesis and is expressed in normal, but not in transformed,hamster (Mesocricetus auratus) oral keratinocytes. Transfection of doc-1cDNA into malignant hamster oral keratinocytes at least partiallyreverts cell morphology, growth rate, and growth properties to thenon-transformed phenotype (Todd, et al., supra). Doc-1 is homologous toTU-166, a gene that is rapidly induced by tumor necrosis factortreatment of apoptosis-resistant mouse fibroblasts (Gordon, H. M. et al.(1992) J. Immunol. 148: 4021-4027).

[0004] Mutations in the tumor suppressor p53 gene are a common featureof many cancers and often appear to be a critical step in thepathogenesis and progression of tumors. Thus it may be possible to usep53 based assays to: 1) screen patients at increased risk for cancer, 2)aid in diagnosis made by traditional methods, and 3) assess theprognosis of individual cancer patients (Chang, F. et al. (1995) J.Clin. Oncol. 13: 1009-1022). In fact, Jacobson, D. R. et al. (1995, Ann.Oncol. 6: S3-S8) report that mutations in tumor suppressor genes, suchas p53, may prove useful in diagnostic tests and screening programs fornon-small-cell lung cancer. The introduction of p53 into cervical cancercells via an adenoviral vector is being investigated as an experimentaltherapy for cervical cancer (Hamada, et al., supra). Such tumorsuppressors are likely to have utility in diagnostics and therapeuticsfor a variety of cancers.

[0005] Cancer remains a major public health concern. Oral squamous cellcarcinoma, for example, is diagnosed in over a half of a million peopleeach year (Vokes, E.E. et al. (1993) N. Engl. J. Med. 328: 184-194).About one half of oral carcinoma patients die within 5 years ofdiagnosis (Silverman, S. Jr. et al. (1990) J. Am. Dent. Assoc. 120:495-499). Although p53 holds promise in the diagnosis and treatment ofmany cancers, it is not universally mutated or overexpressed in cancercells. Furthermore, additional suppressors could prove to be morereadily adaptable to therapeutic use than p53.

[0006] Discovery of proteins related to human tumor suppressors and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions useful in diagnosis, prevention, and treatment ofcancer, such as oral carcinoma.

SUMMARY OF THE INVENTION

[0007] The present invention features a novel human tumor suppressorhereinafter designated TUPRO-2 and characterized as having similarity toDoc-1 from M. auratus.

[0008] Accordingly, the invention features a substantially purifiedTUPRO-2 having the amino acid sequence shown in SEQ ID NO:1.

[0009] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode TUPRO-2. In a particular aspect,the polynucleotide is the nucleotide sequence of SEQ ID NO:2.

[0010] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO:2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2.

[0011] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode TUPRO-2.The present invention also features antibodies which bind specificallyto TUPRO-2, and pharmaceutical compositions comprising substantiallypurified TUPRO-2. The invention also features agonists of TUPRO-2 andthe use thereof for treating cancer. The invention also features amethod for producing TUPRO-2, and methods for treating cancer byadministering TUPRO-2 or a fragment or derivative thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 shows the amino acid sequence (SEQ ID NO:1) and nucleicacid sequence (SEQ ID NO:2) of TUPRO-2. The alignment was produced usingMAcDNASIS PRO software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

[0013]FIG. 2 shows the amino acid sequence alignments between TUPRO-2(SEQ ID NO:1) and M. auratus Doc-1 (GI 606837; SEQ ID NO:3) producedusing the multisequence alignment program of DNASTAR software (DNAStarInc, Madison Wis.).

[0014]FIG. 3 shows the hydrophobicity plot (MAcDNASIS PRO software) forTUPRO-2, SEQ ID NO:1; the positive X axis reflects amino acid position,and the negative Y axis, hydrophobicity.

[0015]FIG. 4 shows the hydrophobicity plot for M. auratus Doc-l, SEQ IDNO:3.

DESCRIPTION OF THE INVENTION

[0016] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0017] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0018] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0019] Definitions

[0020] “Nucleic acid sequence”, as used herein, refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0021] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. “Peptidenucleic acid”, as used herein, refers to a molecule which comprises anoligomer to which an amino acid residue, such as lysine, and an aminogroup have been added. These small molecules, also designated anti-geneagents, stop transcript elongation by binding to their complementarystrand of nucleic acid (Nielsen, P. E. et al. (1993) Anticancer DrugDes. 8:53-63).

[0022] TUPRO-2, as used herein, refers to the amino acid sequences ofsubstantially purified TUPRO-2 obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant. “Consensus”, as used herein, refers to anucleic acid sequence which has been resequenced to resolve uncalledbases, or which has been extended using XL-PCR (Perkin Elmer, Norwalk,Conn.) in the 5′ and/or the 3′ direction and resequenced, or which hasbeen assembled from the overlapping sequences of more than one Incyteclone using the GELVIEW fragment assembly system (GCG, Madison, Wis.),or which has been both extended and assembled.

[0023] A “variant” of TUPRO-2, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0024] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0025] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0026] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0027] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic TUPRO-2, orany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0028] The term “agonist”, as used herein, refers to a molecule which,when bound to TUPRO-2, causes a change in TUPRO-2 which modulates theactivity of TUPRO-2. Agonists may include proteins, nucleic acids,carbohydrates, or any other molecules which bind to TUPRO-2.

[0029] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to TUPRO-2, blocks or modulates thebiological or immunological activity of TUPRO-2. Antagonists andinhibitors may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to TUPRO-2.

[0030] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of TUPRO-2. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of TUPRO-2.

[0031] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure ofTUPRO-2 or portions thereof and, as such, is able to effect some or allof the actions of tumor suppressor-like molecules.

[0032] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding TUPRO-2 or the encoded TUPRO-2.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0033] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated. “Amplification”, as usedherein, refers to the production of additional copies of a nucleic acidsequence and is generally carried out using polymerase chain reaction(PCR) technologies well known in the art (Dieffenbach, C. W. and G. S.Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y.).

[0034] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0035] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0036] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

[0037] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0038] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0039] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0040] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0041] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human TUPRO-2 and fragments thereof.

[0042] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0043] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0044] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0045] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encodingTUPRO-2 or fragments thereof may comprise a cell, chromosomes isolatedfrom a cell (e.g., a spread of metaphase chromosomes), genomic DNA (insolution or bound to a solid support such as for Southern analysis), RNA(in solution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0046] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding TUPRO-2 in a sample and therebycorrelates with expression of the transcript from the polynucleotideencoding the protein.

[0047] “Alterations” in the polynucleotide of SEQ ID NO:2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding TUPRO-2 including deletions, insertions, and point mutationsthat may be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes TUPRO-2 (e.g., by alterations in the pattern ofrestriction fragment length polymorphisms capable of hybridizing to SEQID NO:2), the inability of a selected fragment of SEQ ID NO:2 tohybridize to a sample of genomic DNA (e.g., using allele-specificoligonucleotide probes), and improper or unexpected hybridization, suchas hybridization to a locus other than the normal chromosomal locus forthe polynucleotide sequence encoding TUPRO-2 (e.g., using fluorescent insitu hybridization [FISH] to metaphase chromosome spreads).

[0048] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bindTUPRO-2 polypeptides can be prepared using intact polypeptides orfragments containing small peptides of interest as the immunizingantigen. The polypeptide or peptide used to immunize an animal can bederived from the transition of RNA or synthesized chemically, and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0049] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0050] The Invention

[0051] The invention is based on the discovery of a novel human tumorsuppressor, (TUPRO-2), the polynucleotides encoding TUPRO-2, and the useof these compositions for the diagnosis, prevention, or treatment ofcancer.

[0052] Nucleic acids encoding the human TUPRO-2 of the present inventionwere first identified in Incyte Clone 2446131 from the THP-1pro-monocyte cell cDNA library (THP1NOT03) through a computer-generatedsearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the following overlapping and/or extended nucleicacid sequences: Incyte Clones 2446131 (THP1NOT03), 730617 (LUNGNOT03),1998850 (BRSTTUT03), 2158648 (BRAINOT09), and 2277445 (PROSNON01).

[0053] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIG. 1.TUPRO-2 is 126 amino acids in length. TUPRO-2 has chemical andstructural homology with M. auratus Doc-1 (GI 606837; SEQ ID NO:3). Inparticular, TUPRO-2 and M. auratus Doc-1 share 54% identity. Asillustrated by FIGS. 3 and 4, TUPRO-2 and M. auratus Doc-1 have rathersimilar hydrophobicity plots. Northern analysis revealed the expressionof this sequence in various cDNA libraries, a high proportion of whichare derived from cancer patients.

[0054] The invention also encompasses TUPRO-2 variants. A preferredTUPRO-2 variant is one having at least 80%, and more preferably 90%,amino acid sequence identity to the TUPRO-2 amino acid sequence (SEQ IDNO:1). A most preferred TUPRO-2 variant is one having at least 95% aminoacid sequence identity to SEQ ID NO:1.

[0055] The invention also encompasses polynucleotides which encodeTUPRO-2. Accordingly, any nucleic acid sequence which encodes the aminoacid sequence of TUPRO-2 can be used to generate recombinant moleculeswhich express TUPRO-2. In a particular embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequence ofSEQ ID NO:2 as shown in FIG. 1.

[0056] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding TUPRO-2, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring TUPRO-2, and all suchvariations are to be considered as being specifically disclosed.

[0057] Although nucleotide sequences which encode TUPRO-2 and itsvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring TUPRO-2 under appropriately selectedconditions of stringency, it may be advantageous to produce nucleotidesequences encoding TUPRO-2 or its derivatives possessing a substantiallydifferent codon usage. Codons may be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding TUPRO-2 and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0058] The invention also encompasses production of DNA sequences, orportions thereof, which encode TUPRO-2 and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding TUPRO-2 or any portionthereof.

[0059] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0060] Altered nucleic acid sequences encoding TUPRO-2 which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent TUPRO-2. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent TUPRO-2. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of TUPRO-2 is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0061] Also included within the scope of the present invention arealleles of the genes encoding TUPRO-2. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0062] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, OH), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE amplification systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the MICROLAB 2200 liquid transfer system(Hamilton, Reno, Nev.), the PTC200 thermal cycler (MJ Research,Watertown, Mass.) and the ABI 377 DNA sequencer (Perkin Elmer).

[0063] The nucleic acid sequences encoding TUPRO-2 may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0064] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 software (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0065] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1: 111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0066] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0067] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0068] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0069] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode TUPRO-2, or fusion proteins orfunctional equivalents thereof, may be used in recombinant DNA moleculesto direct expression of TUPRO-2 in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences whichencode substantially the same or a functionally equivalent amino acidsequence may be produced and these sequences may be used to clone andexpress TUPRO-2.

[0070] As will be understood by those of skill in the art, it may beadvantageous to produce TUPRO-2-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0071] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterTUPRO-2 encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0072] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding TUPRO-2 may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of TUPRO-2 activity, it may be usefulto encode a chimeric TUPRO-2 protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the TUPRO-2 encoding sequenceand the heterologous protein sequence, so that TUPRO-2 may be cleavedand purified away from the heterologous moiety.

[0073] In another embodiment, sequences encoding TUPRO-2 may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of TUPRO-2, or a portion thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431 A peptide synthesizer (Perkin Elmer).

[0074] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of TUPRO-2, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0075] In order to express a biologically active TUPRO-2, the nucleotidesequences encoding TUPRO-2 or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0076] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encodingTUPRO-2 and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook, J. et al. (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.

[0077] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding TUPRO-2. These include, but arenot limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems.

[0078] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORTl plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encodingTUPRO-2, vectors based on SV40 or EBV may be used with an appropriateselectable marker.

[0079] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for TUPRO-2. For example, whenlarge quantities of TUPRO-2 are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding TUPRO-2 may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0080] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0081] In cases where plant expression vectors are used, the expressionof sequences encoding TUPRO-2 may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, NY; pp. 191-196).

[0082] An insect system may also be used to express TUPRO-2. Forexample, in one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encodingTUPRO-2 may be cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of TUPRO-2 will render the polyhedringene inactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which TUPRO-2 may beexpressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.91:3224-3227).

[0083] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding TUPRO-2 may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing TUPRO-2 in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0084] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding TUPRO-2. Such signalsinclude the ATG initiation codon and adjacent sequences. In cases wheresequences encoding TUPRO-2, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0085] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0086] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress TUPRO-2 may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0087] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0088] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding TUPRO-2is inserted within a marker gene sequence, recombinant cells containingsequences encoding TUPRO-2 can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding TUPRO-2 under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0089] Alternatively, host cells which contain the nucleic acid sequenceencoding TUPRO-2 and express TUPRO-2 may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0090] The presence of polynucleotide sequences encoding TUPRO-2 can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding TUPRO-2.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding TUPRO-2 todetect transformants containing DNA or RNA encoding TUPRO-2. As usedherein “oligonucleotides” or “oligomers” refer to a nucleic acidsequence of at least about 10 nucleotides and as many as about 60nucleotides, preferably about 15 to 30 nucleotides, and more preferablyabout 20-25 nucleotides, which can be used as a probe or amplimer.

[0091] A variety of protocols for detecting and measuring the expressionof TUPRO-2, using either polyclonal or monoclonal antibodies specificfor the protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TUPRO-2 is preferred, but a competitive binding assay may beemployed. These and other assays are described, among other places, inHampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APSPress, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:121 1-1216).

[0092] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encodingTUPRO-2 include oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding TUPRO-2, or any portions thereof may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used,include radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0093] Host cells transformed with nucleotide sequences encoding TUPRO-2may be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode TUPRO-2 may be designed to contain signal sequences which directsecretion of TUPRO-2 through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingTUPRO-2 to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and TUPRO-2 may be used to facilitate purification.One such expression vector provides for expression of a fusion proteincontaining TUPRO-2 and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography) as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying TUPRO-2 from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

[0094] In addition to recombinant production, fragments of TUPRO-2 maybe produced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 43 1APeptide Synthesizer (Perkin Elmer). Various fragments of TUPRO-2 may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0095] Therapeutics

[0096] Based on the chemical and structural homology that exists betweenTUPRO-2 and M. auratus Doc-1, and the expression of TUPRO-2 in tissuesderived from cancer patients, TUPRO-2 appears to be associated with thedevelopment of cancer.

[0097] Mutations in tumor suppressor genes are often found in humantumor cells and in many instances are thought to be critical to both theinitiation of tumor development and to the tumor's ability to survivechemotherapy. Therefore, in one embodiment, TUPRO-2 or a fragment orderivative thereof may be administered to a subject to treat or preventcancer. Types of cancer include, but are not limited to, adenocarcinoma;leukemia; melanoma; lymphoma; sarcoma; and cancers of the colon, liver,brain, small intestine, large intestine, breast, ovary, kidney, lung,and prostate.

[0098] In another embodiment, a vector capable of expressing TUPRO-2, ora fragment or a derivative thereof, may also be administered to asubject to treat or prevent cancer including, but not limited, the typesof cancers listed above.

[0099] In another embodiment, agonists which are specific for TUPRO-2may be used to stimulate or prolong the activity of TUPRO-2 and may beadministered to a subject to treat or prevent cancers including, but notlimited to, the cancers listed above.

[0100] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, antisense sequences or vectorsdescribed above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0101] Antagonists or inhibitors of TUPRO-2 may be produced usingmethods which are generally known in the art. In particular, purifiedTUPRO-2 may be used to produce antibodies or to screen libraries ofpharmaceutical agents to identify those which specifically bind TUPRO-2.

[0102] The antibodies may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0103] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith TUPRO-2 or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0104] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to TUPRO-2 have an amino acid sequenceconsisting of at least five amino acids, and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Short stretches of TUPRO-2 amino acids may be fused with those ofanother protein such as keyhole limpet hemocyanin and antibody producedagainst the chimeric molecule.

[0105] Monoclonal antibodies to TUPRO-2 may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0106] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceTUPRO-2-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0107] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0108] Antibody fragments which contain specific binding sites forTUPRO-2 may also be generated. For example, such fragments include, butare not limited to, the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0109] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between TUPRO-2 and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering TUPRO-2 epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0110] In another embodiment of the invention, the polynucleotidesencoding TUPRO-2, or any fragment thereof, or antisense molecules, maybe used for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding TUPRO-2 may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding TUPRO-2. Thus, antisense molecules may be usedto modulate TUPRO-2 activity, or to achieve regulation of gene function.Such technology is now well known in the art, and sense or antisenseoligomers or larger fragments, can be designed from various locationsalong the coding or control regions of sequences encoding TUPRO-2.

[0111] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingTUPRO-2. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

[0112] Genes encoding TUPRO-2 can be turned off by transforming a cellor tissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes TUPRO-2. Suchconstructs may be used to introduce untranslatable sense or antisensesequences into a cell. Even in the absence of integration into the DNA,such vectors may continue to transcribe RNA molecules until they aredisabled by endogenous nucleases. Transient expression may last for amonth or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

[0113] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding TUPRO-2, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions -10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0114] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding TUPRO-2.

[0115] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0116] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding TUPRO-2. Such DNA sequences maybe incorporated into a wide variety of vectors with suitable RNApolymerase promoters such as T7 or SP6. Alternatively, these cDNAconstructs that synthesize antisense RNA constitutively or inducibly canbe introduced into cell lines, cells, or tissues.

[0117] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0118] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0119] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0120] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist ofTUPRO-2, antibodies to TUPRO-2, mimetics, agonists, antagonists, orinhibitors of TUPRO-2. The compositions may be administered alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. The compositions may be administered to a patientalone, or in combination with other agents, drugs or hormones.

[0121] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0122] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0123] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0124] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0125] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0126] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0127] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0128] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0129] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0130] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0131] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of TUPRO-2, such labeling wouldinclude amount, frequency, and method of administration.

[0132] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0133] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0134] A therapeutically effective dose refers to that amount of activeingredient, for example TUPRO-2 or fragments thereof, antibodies ofTUPRO-2, agonists, antagonists or inhibitors of TUPRO-2, whichameliorates the symptoms or condition. Therapeutic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., ED50 (the dose therapeutically effectivein 50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.

[0135] Pharmaceutical compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosages for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED50 with little or notoxicity. The dosage varies within 'this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0136] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0137] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0138] Diagnostics

[0139] In another embodiment, antibodies which specifically bind TUPRO-2may be used for the diagnosis of conditions or diseases characterized byexpression of TUPRO-2, or in assays to monitor patients being treatedwith TUPRO-2, agonists, antagonists or inhibitors. The antibodies usefulfor diagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for TUPRO-2 includemethods which utilize the antibody and a label to detect TUPRO-2 inhuman body fluids or extracts of cells or tissues. The antibodies may beused with or without modification, and may be labeled by joining them,either covalently or non-covalently, with a reporter molecule. A widevariety of reporter molecules which are known in the art may be used,several of which are described above.

[0140] A variety of protocols including ELISA, RIA, and FACS formeasuring TUPRO-2 are known in the art and provide a basis fordiagnosing altered or abnormal levels of TUPRO-2 expression. Normal orstandard values for TUPRO-2 expression are established by combining bodyfluids or cell extracts taken from normal mammalian subjects, preferablyhuman, with antibody to TUPRO-2 under conditions suitable for complexformation. The amount of standard complex formation may be quantified byvarious methods, but preferably by photometric means. Quantities ofTUPRO-2 expressed in subject, control, and disease samples from biopsiedtissues are compared with the standard values. Deviation betweenstandard and subject values establishes the parameters for diagnosingdisease.

[0141] In another embodiment of the invention, the polynucleotidesencoding TUPRO-2 may be used for diagnostic purposes. Thepolynucleotides which may be used include oligonucleotide sequences,antisense RNA and DNA molecules, and PNAs. The polynucleotides may beused to detect and quantitate gene expression in biopsied tissues inwhich expression of TUPRO-2 may be correlated with disease. Thediagnostic assay may be used to distinguish between absence, presence,and excess expression of TUPRO-2, and to monitor regulation of TUPRO-2levels during therapeutic intervention.

[0142] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TUPRO-2 or closely related molecules, may be used to identifynucleic acid sequences which encode TUPRO-2. The specificity of theprobe, whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding TUPRO-2, alleles, or related sequences.

[0143] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the TUPRO-2 encoding sequences. The hybridization probes of thesubject invention may be DNA or RNA and derived from the nucleotidesequence of SEQ ID NO:2 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring TUPRO-2.

[0144] Means for producing specific hybridization probes for DNAsencoding TUPRO-2 include the cloning of nucleic acid sequences encodingTUPRO-2 or TUPRO-2 derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as 32P or 35S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0145] Polynucleotide sequences encoding TUPRO-2 may be used for thediagnosis of conditions or diseases which are associated with expressionof TUPRO-2. Examples of such conditions or diseases include cancers ofthe stomach, lung, small intestine, brain, liver, ovaries, breast,testes, thyroid, large intestine, colon, pancreas, heart, and prostate;ulcerative colitis; and Alzheimer's disease. The polynucleotidesequences encoding TUPRO-2 may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dip stick, pIN, ELISA or chip assays utilizing fluids or tissues frompatient biopsies to detect altered TUPRO-2 expression. Such qualitativeor quantitative methods are well known in the art.

[0146] In a particular aspect, the nucleotide sequences encoding TUPRO-2may be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding TUPRO-2 may be labeled by standard methods, and added to afluid or tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding TUPRO-2 in the sample indicates thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0147] In order to provide a basis for the diagnosis of diseaseassociated with expression of TUPRO-2, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes TUPRO-2,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0148] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0149] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0150] Additional diagnostic uses for oligonucleotides designed from thesequences encoding TUPRO-2 may involve the use of PCR. Such oligomersmay be chemically synthesized, generated enzymatically, or produced froma recombinant source. Oligomers will preferably consist of twonucleotide sequences, one with sense orientation (5′→3′) and anotherwith antisense (3′→5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0151] Methods which may also be used to quantitate the expression ofTUPRO-2 include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P.C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0152] In another embodiment of the invention, the nucleic acidsequences which encode TUPRO-2 may also be used to generatehybridization probes which are useful for mapping the naturallyoccurring genomic sequence. The sequences may be mapped to a particularchromosome or to a specific region of the chromosome using well knowntechniques . Such techniques include FISH, FACS, or artificialchromosome constructions, such as yeast artificial chromosomes,bacterial artificial chromosomes, bacterial PI constructions or singlechromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood Rev.7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

[0153] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:198 1f). Correlation between the location of thegene encoding TUPRO-2 on a physical chromosomal map and a specificdisease , or predisposition to a specific disease, may help delimit theregion of DNA associated with that genetic disease. The nucleotidesequences of the subject invention may be used to detect differences ingene sequences between normal, carrier, or affected individuals.

[0154] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0155] In another embodiment of the invention, TUPRO-2, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenTUPRO-2 and the agent being tested, may be measured.

[0156] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to TUPRO-2 largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with TUPRO-2, or fragments thereof, and washed.Bound TUPRO-2 is then detected by methods well known in the art.Purified TUPRO-2 can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0157] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding TUPRO-2specifically compete with a test compound for binding TUPRO-2. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with TUPRO-2.

[0158] In additional embodiments, the nucleotide sequences which encodeTUPRO-2 may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0159] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0160] I THP1NOT03 cDNA Library Construction

[0161] The THP1NOT03 library was constructed using 1 microgram of polyARNA isolated from untreated THP-1 cells. THP-1 (ATCC TIB 202) is a humanpromonocyte line derived from the peripheral blood of a 1-year-oldCaucasian male with acute monocytic leukemia. The THP-1 cells werehomogenized and lysed using a Brinkmann PT-3000 homogenizer (BrinkmannInstruments, Westbury, N.J.) in guanidinium isothiocyanate solution. Thelysate was centrifuged over a 5.7 M CsCl cushion using a Beckman SW28rotor in a Beckman L8-70M ultracentrifuge (Beckman Instruments) for 18hours at 25,000 rpm at ambient temperature. The RNA was extracted withacid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5volumes of ethanol, resuspended in RNAse-free water, and DNase treatedat 37° C. Extraction and precipitation were repeated as before. The mRNAwas then isolated with the QIAGEN OLIGOTEX kit (QIAGEN, Inc.,Chatsworth, Calif.) and used to construct the cDNA library.

[0162] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning(Gaithersburg, Md.). cDNAs were fractionated on a SEPHAROSE CL4B column(Pharmacia), and those cDNAs exceeding 400 bp were ligated intoPSPORT 1. The plasmid PSPORT1 was subsequently transformed into DH5αcompetent cells (Gibco/BRL).

[0163] II Isolation and Sequencing of cDNA Clones

[0164] Plasmid DNA was released from the cells and purified using theR.E.A.L. PREP 96 plasmid kit (QIAGEN, Inc.). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (LIFE TECHNOLOGIES) with carbenicillin at 25mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0165] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94:44 1f), using a MICROLAB 2200 liquid transfer system(Hamilton, Reno, Nev.) in combination with the PTC200 thermal cycler(PTC200 from MJ Research, Watertown, Mass.) and 377 DNA sequencingsystem (Applied Biosystems); and the reading frame was determined.

[0166] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0167] The nucleotide sequences of the Sequence Listing as well as theamino acid sequences deduced from them were used as query sequencesagainst databases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases, which contain previously identified and annotated sequences,were searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul (1993) supra,Altschul (1990) supra).

[0168] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms such as the one described in Smith et al. (1992, ProteinEngineering 5:35-51), incorporated herein by reference, could have beenused when dealing with primary sequence patterns and secondary structuregap penalties. The sequences disclosed in this application have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

[0169] The BLAST approach, as detailed in Karlin et al. (supra) andincorporated herein by reference, searched for matches between a querysequence and a database sequence. BLAST evaluated the statisticalsignificance of any matches found, and reported only those matches thatsatisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

[0170] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam); and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology. The relevantdatabase for a particular match were reported as Glxxx±p (where xxx ispri, rod, etc., and if present, p=peptide). The product score iscalculated as follows: the % nucleotide or amino acid identity [betweenthe query and reference sequences] in BLAST is multiplied by the %maximum possible BLAST score [based on the lengths of query andreference sequences] and then divided by 100. Where an Incyte Clone washomologous to several sequences, up to five matches were provided withtheir relevant scores. In an analogy to the hybridization proceduresused in the laboratory, the electronic stringency for an exact match wasset at 70, and the conservative lower limit for an exact match was setat approximately 40 (with 1-2% error due to uncalled bases).

[0171] IV Northern Analysis

[0172] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0173] Analogous computer techniques using BLAST (Altschul, S.F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0174] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \quad \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0175] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0176] The results of northern analysis are reported as a list oflibraries in which the transcript encoding TUPRO-2 occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0177] V Extension of TUPRO-2-encoding Polynucleotides

[0178] Nucleic acid sequence of Incyte clone 2366261 or SEQ ID NO:2 isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 software (National Biosciences),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68 °-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0179] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0180] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the PTC200 thermal cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (and holding)

[0181] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0182] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

[0183] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0184] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0185] VI Labeling and Use of Hybridization Probes

[0186] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with Sephadex G-25superfine resin column (Pharmacia & Upjohn). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN).

[0187] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (NYTRAN PLUS, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0188] VII Antisense Molecules

[0189] Antisense molecules or the complement of the TUPRO-2-encodingsequence, or any part thereof, is used to inhibit in vivo or in vitroexpression of naturally occurring TUPRO-2. Although use of antisenseoligonucleotides, comprising about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. An oligonucleotide based on the coding sequences of TUPRO-2,as shown in FIG. 1, is used to inhibit expression of naturally occurringTUPRO-2. The complementary oligonucleotide is designed from the mostunique 5′ sequence as shown in FIG. 1 and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of an TUPRO-2-encoding transcriptby preventing the ribosome from binding. Using an appropriate portion ofthe signal and 5′ sequence of SEQ ID NO:2, an effective antisenseoligonucleotide includes any 15-20 nucleotides spanning the region whichtranslates into the signal or 5′ coding sequence of the polypeptide asshown in FIG. 1.

[0190] VIII Expression of TUPRO-2

[0191] Expression of TUPRO-2 is accomplished by subcloning the cDNAsinto appropriate vectors and transforming the vectors into host cells.In this case, the cloning vector, PSPORT1, previously used for thegeneration of the cDNA library is used to express TUPRO-2 in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0192] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of TUPRO-2 into the bacterial growth media which can be useddirectly in the following assay for activity. IX Demonstration ofTUPRO-2 Activity

[0193] TUPRO-2 activity can be measured by a method described by Todd etal. (supra). The TUPRO-2 gene is placed on a mammalian expression vectorand transfected into malignant HCPC-1 hamster oral keratinocytes.Transfected cells are seeded at 4×10³ cells in 35 mm dishes in DMEM/1%Agar. Cultures are incubated 7 days at 37° C. in a CO₂ incubator.Staining with crystal violet reveals colonies. TUPRO-2 tumor suppressoractivity is assessed by ELISA and by recording the number of coloniesrelative to controls.

[0194] X Production of TUPRO-2 Specific Antibodies

[0195] TUPRO-2 that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0196] Typically, the oligopeptides are 15 residues in length,synthesized using an ABI 43 1A peptide synthesizer (Applied Biosystems)using fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0197] XI Purification of Naturally Occurring TUPRO-2 Using SpecificAntibodies

[0198] Naturally occurring or recombinant TUPRO-2 is substantiallypurified by immunoaffinity chromatography using antibodies specific forTUPRO-2. An immunoaffinity column is constructed by covalently couplingTUPRO-2 antibody to an activated chromatographic resin, such asCnBr-activated SEPHAROSE (Pharmacia & Upjohn). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0199] Media containing TUPRO-2 is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of TUPRO-2 (e.g., high ionic strength buffers inthe presence of detergent). The column is eluted under conditions thatdisrupt antibody/TUPRO-2 binding (eg, a buffer of pH 2-3 or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andTUPRO-2 is collected.

[0200] XII Identification of Molecules Which Interact with TUPRO-2

[0201] TUPRO-2 or biologically active fragments thereof are labeled with1251 Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled TUPRO-2, washed and any wells withlabeled TUPRO-2 complex are assayed. Data obtained using differentconcentrations of TUPRO-2 are used to calculate values for the number,affinity, and association of TUPRO-2 with the candidate molecules.

[0202] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 3 126 amino acids amino acid single linear THPINOT03 2446131 1 Met SerTyr Lys Pro Ile Ala Pro Ala Pro Ser Xaa Thr Pro Gly Ser 1 5 10 15 SerThr Pro Gly Pro Gly Thr Pro Val Pro Thr Gly Ser Val Pro Ser 20 25 30 ProSer Gly Ser Val Pro Gly Ala Gly Ala Pro Phe Arg Pro Leu Phe 35 40 45 AsnAsp Phe Gly Pro Pro Ser Met Gly Tyr Val Gln Ala Met Lys Pro 50 55 60 ProGly Ala Gln Gly Ser Gln Ser Thr Tyr Thr Asp Leu Leu Ser Val 65 70 75 80Ile Glu Glu Met Gly Lys Glu Ile Arg Pro Thr Tyr Ala Gly Ser Lys 85 90 95Ser Ala Met Glu Arg Leu Lys Arg Gly Ile Ile His Ala Arg Ala Leu 100 105110 Val Arg Glu Cys Leu Ala Glu Thr Glu Arg Asn Ala Arg Thr 115 120 125667 base pairs nucleic acid single linear THPINOT03 2446131 2 GGCTGAGCGGCCCCGCAGCC AACCCCCGAG GAGCGGCCGG CTGGCGTCCG CCGCGCCCAG 60 GAGTTGGGGATGTCCTACAA ACCCATCGCC CCTGCTCCCA GCAKCACCCC TGGCTCCAGC 120 ACCCCTGGGCCGGGCACCCC GGTCCCTACA GGAAGCGTCC CGTCGCCGTC GGGCTCAGTG 180 CCAGGAGCCGGCGCTCCTTT CAGACCGCTG TTTAACGACT TTGGACCGCC TTCCATGGGC 240 TACGTGCAGGCGATGAAGCC ACCCGGCGCC CAGGGCTCCC AGAGCACCTA CACGGACCTG 300 CTGTCAGTCATAGAGGAGAT GGGCAAAGAG ATCCGGCCTA CCTATGCTGG CAGCAAGAGC 360 GCCATGGAGCGCCTGAAGAG AGGTATCATC CATGCCCGGG CCCTAGTCAG AGAGTGCCTG 420 GCAGAGACAGAGCGGAACGC CCGCACGTAA CAGGAAGCGC CTCGGCCTCA GCGTCTGGAC 480 CTATCCGGCCACTGCAGAGC ACCCGCTTCT CCCTGGCCTT CATCCCGAGT TGCACTAACC 540 ATCCTGGGCTTCCTGTCCTG TGTCCCTTGG TGGGTCCCCT CCAGGAACCA AGGAGTGGCC 600 CTCCAGGTGGCAGCACTAAG GACACCCCCC CACAACAAGA GTTAGCAGCG AGGTCCCCAT 660 GAGTCCC 667114 amino acids amino acid single linear GenBank 606837 3 Met Ser TyrLys Pro Asn Leu Thr Ala His Met Pro Ala Ala Ala Leu 1 5 10 15 Asn AlaGly Ser Val His Ser Pro Ser Thr Ser Met Ala Thr Ser Ser 20 25 30 Gln TyrArg Gln Leu Leu Ser Asp Tyr Gly Pro Pro Ser Leu Gly Tyr 35 40 45 Thr GlnGly Thr Gly Asn Ser Gln Val Pro Gln Ser Lys Tyr Ala Glu 50 55 60 Leu LeuAla Ile Ile Glu Glu Leu Gly Lys Glu Ile Arg Pro Thr Tyr 65 70 75 80 AlaGly Ser Lys Ser Ala Met Glu Arg Leu Lys Arg Gly Ile Ile His 85 90 95 AlaArg Ser Leu Val Arg Glu Cys Leu Ala Glu Thr Glu Arg Asn Ala 100 105 110Arg Ser

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO:1, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence of SEQ IDNO:1, c) a biologically active fragment of a polypeptide having an aminoacid of SEQ ID NO:1, and d) an immunogenic fragment of a polypeptidehaving an amino acid of SEQ ID NO:1.
 2. An isolated polypeptide of claim1 comprising an amino acid sequence of SEQ ID NO:1.
 3. An isolatedpolynucleotide encoding a polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide of claim
 2. 5. An isolatedpolynucleotide of claim 4 comprising a polynucleotide sequence of SEQ IDNO:2.
 6. A recombinant polynucleotide comprising a promoter sequenceoperably linked to a polynucleotide of claim
 3. 7. A cell transformedwith a recombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide has an amino acid sequence of SEQ ID NO:1. 11.An isolated antibody which specifically binds to a polypeptide ofclaim
 1. 12. An isolated polynucleotide selected from the groupconsisting of: a) a polynucleotide comprising a polynucleotide sequenceof SEQ ID NO:2, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence of SEQ ID NO:2, c) a polynucleotide complementary to apolynucleotide of a), d) a polynucleotide complementary to apolynucleotide of b), and e) an RNA equivalent of a)-d).
 13. An isolatedpolynucleotide comprising at least 60 contiguous nucleotides of apolynucleotide of claim
 12. 14. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) hybridizingthe sample with a probe comprising at least 20 contiguous nucleotidescomprising a sequence complementary to said target polynucleotide in thesample, and which probe specifically hybridizes to said targetpolynucleotide, under conditions whereby a hybridization complex isformed between said probe and said target polynucleotide or fragmentsthereof, and b) detecting the presence or absence of said hybridizationcomplex, and, optionally, if present, the amount thereof.
 15. A methodof claim 14, wherein the probe comprises at least 60 contiguousnucleotides.
 16. A method of detecting a target polynucleotide in asample, said target polynucleotide having a sequence of a polynucleotideof claim 12, the method comprising: a) amplifying said targetpolynucleotide or fragment thereof using polymerase chain reactionamplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional TUPRO-2,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional TUPRO-2, comprising administering to a patient in need ofsuch treatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional TUPRO-2, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, themethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of TUPRO-2 in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of TUPRO-2 in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofTUPRO-2 in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide having an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibodies from said animal, and c) screening theisolated antibodies with the polypeptide, thereby identifying apolyclonal antibody which binds specifically to a polypeptide having anamino acid sequence of SEQ ID NO:1.
 37. A polyclonal antibody producedby a method of claim
 36. 38. A composition comprising the polyclonalantibody of claim 37 and a suitable carrier.
 39. A method of making amonoclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence of SEQ ID NO:1, or an immunogenic fragmentthereof, under conditions to elicit an antibody response, b) isolatingantibody producing cells from the animal, c) fusing the antibodyproducing cells with immortalized cells to form monoclonalantibody-producing hybridoma cells, d) culturing the hybridoma cells,and e) isolating from the culture monoclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence of SEQ IDNO:1.
 40. A monoclonal antibody produced by a method of claim
 39. 41. Acomposition comprising the monoclonal antibody of claim 40 and asuitable carrier.
 42. The antibody of claim 11, wherein the antibody isproduced by screening a Fab expression library.
 43. The antibody ofclaim 11, wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptide havingan amino acid sequence of SEQ ID NO:1 in a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having an amino acid sequence ofSEQ ID NO:1 in the sample.
 45. A method of purifying a polypeptidehaving an amino acid sequence of SEQ ID NO:1 from a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:1.
 46. A microarray wherein at least one element of the microarrayis a polynucleotide of claim
 13. 47. A method of generating a transcriptimage of a sample which contains polynucleotides, the method comprising:a) labeling the polynucleotides of the sample, b) contacting theelements of the microarray of claim 46 with the labeled polynucleotidesof the sample under conditions suitable for the formation of ahybridization complex, and c) quantifying the expression of thepolynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.